This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. Recent studies have focused on: 1) analysis of the solution conformation and dynamics of the ribonuclease H (RNase H) domain of HIV reverse transcriptase, and the characterization of substrate-induced active site formation; 2) understanding the protein-mediated interactions of RT substrates and inhibitors; 3) understanding the complex conformational maturation process that converts the initiall p66 protein into the p66/p66' homodimer, and finally the p66/p51 heterodimer; 4) understanding the basis for the subunit-dependent elimination of one RH domain. Project 1. Maturation pathway of HIV-1 reverse transcriptase. HIV reverse transcriptase (RT) plays a multifunctional role in the transformation of viral RNA into dsDNA and represents a primary target for treatment of AIDS. Currently, all of the drugs in clinical use target the mature RT p66/p51 heterodimer, however, a single p66 peptide chain functions as the precursor for each subunit of the RT heterodimer, requiring a complex maturation process that includes subunit-selective elimination of a single ribonuclease H (RH) domain. The need for such a process is a consequence of a metamorphic polymerase domain that is able to adopt different structures in each RT subunit, allowing it to fulfill two different functional roles. The metamorphic polymerase domain reduces the need for additional coding sequences in the HIV gene, consistent with evolutionary pressures on the size of the RNA viral genome, while requiring a more complex structural maturation process. Hypotheses for the formation and maturation of the RT homodimer include proposals in which RH domain proteolysis precedes heterodimer formation, models in which p66 forms an initially symmetric homodimer followed by RH domain unfolding leading to an asymmetric homodimer, and models in which an initially formed asymmetric homodimer leads to partial RH domain unfolding. Until recently, no detailed structural data were available for the p66 monomer and very little structural evidence was available to support or refute any of the above models. Not only does this represent a significant gap in understanding the behavior of an important viral enzyme but also the intermediates involved in heterodimer formation provide potentially useful targets for the development of new interventional strategies. In order to more fully characterize the transformation from monomer to mature heterodimer and to resolve conflicting hypotheses, we utilized site-directed mutagenesis to assign the isoleucine &#948;-methyl resonances arising from the connection domains, allowing a more complete description of the changes taking place in this highly plastic region of the protein. This information also provided insight into the coordinated changes that link conformational maturation of the p66' connection' domain to RH' unfolding. We also performed molecular dynamics simulations for some of the early isomerization events not directly accessible to our NMR measurements. Using our recently introduced isomerization-restricted p66 mutant, we also demonstrated subunit-selective labeling, which allows us to the study the conformational maturation of the p66' subunit of RT without additional resonances from the p66 subunit, greatly reducing the resonance overlap problem. These studies have provided a more complete description of the complex conformational maturation processes leading to formation of the p66/p51 RT heterodimer. Starting from an inactive conformation, the p66 precursor undergoes a unimolecular isomerization to a structure similar to its active form, exposing a large hydrophobic surface that facilitates initial homodimer formation. The resulting p66/p66 homodimer exists as a structural heterodimer, after which a series of conformational adjustments on different time scales can be observed. Formation of the inter-subunit RH:thumb interface occurs at a relatively early stage, while maturation of the connection and unfolding of the RH domains are linked and occur on a much slower time scale, with time constant 7 h. Project 2. According to our recent studies, p66 monomers are able to form structurally asymmetric p66/p66' homodimers that contains two, folded RH domains. The RH' domain on p66' is then subject to selective destabilization as a result of the transfer of residues from RH' to the polymerase' domain on p66'. In isolation, the RH domain of RT has been reported to exist both as a monomer and a domain-swapped dimer. Since formation of the domain swapped dimer requires a substantial degree of unfolding, this observation is consistent with our previous analysis. In order to more fully understand the nature of this unfolding process and to determine what intrinsic factors facilitate this unfolding pathway, we have investigated the structure of the domain swapped RH domain in detail. These studies indicate the existence of several intrinsically destabilizing features of the RH domain, particularly substantial strain in the B-D loop connecting helices B and D, and orientational instability of helix B that results from a lack of side chain hydrogen bond interactions. Further, structural analysis indicates that the N-terminal tyrosine residue, Tyr427, plays an important role in mediating the interaction between helices B and D. Thus, the intrinsic instability of the domain produces instability in the Tyr427 binding pocket that facilitates its release. Conversely, removal of Tyr427 from its binding pocket further destabilizes the structure by eliminating an important component of the helix B helix D interaction. Further supporting the analysis based on structural studies, we characterized the hydrogen/deuterium exchange behavior of an RH construct in which Tyr427 and Gln428 are not present (RH&#8710;NT). Based on NMR data, this construct adopts a structure very similar to that of the full RH domain, however as judged by H/D exchange, it is much less stable. Conversion of the domain swapped dimer into the monomer form is accelerated by 300-fold in the RH&#8710;NT construct, indicating that the unfolding pathway is dramatically facilitated. These results are consistent with the tug-of-war model in which the polymerase and RH domains compete for Tyr427 and nearby residues on one of the homodimer subunits.